Influenza A virus is the causative agent of flu and is responsible for several thousand deaths annually. There is also the risk of pandemic outbreaks occurring again and again in the future. With high level of resistance to current antiviral drugs among the circulating influenza A viruses, it is important that new classes of drugs are discovered that target other fundamental steps in the life cycle of the virus. Influenza virus depends on the host translational machinery to produce viral proteins in infected cells. Previous studies have indicated that influenza virus is able to up-regulate the production of viral proteins in infected cells with the help of Non-Structural Protein 1 (NS1). NS1 is a 26 kDa viral protein that binds to double-stranded and single- stranded RNAs. NS1 has two functional domains: RNA-binding domain (RBD) and Effector Domain (ED). The primary function of NS1 is to reduce the interferon-? response in cells by interacting with several host proteins. Additionally, NS1 has been proposed to stimulate the translation of viral mRNAs by interacting with Poly (A) Binding Protein 1 (PABP1) and eukaryotic Initiation Factor 4G (eIF4G). However, the mechanism used by NS1 to specifically stimulating the translation of viral mRNAs is not clear. We propose to use new, fluorescence-based quantitative methods to analyze the interaction of NS1 with PABP1, eIF4G, and viral RNA sequences. In preliminary studies, we have determined the equilibrium dissociation constant (KD) for NS1 and PABP1 binding to several RNAs using fluorescence anisotropy. As expected, PABP1 binds with high binding affinity to poly(A) RNA, but not to poly(C) RNA. Interestingly, our studies show that NS1 binds to a double- stranded RNA but not to conserved single stranded RNA sequences from the 5'-untranslated region (5'-UTR) of viral mRNAs. We plan to identify the RNA motifs in viral mRNAs that are recognized by NS1 using a combination of quantitative binding assays and RNA chemical probing experiments. Additionally, the interaction of NS1 with PABP1 and eIF4G will be analyzed using Frster Resonance Energy Transfer (FRET) assays. These studies will reveal whether NS1 binds to specific RNA motifs to recruit PABP1 and eIF4G to stimulate the translation of viral mRNAs. Understanding the mechanism used by NS1 to stimulate viral mRNA translation will provide new avenues to prevent infection or lower the severity of the disease.